The [4Fe‐4S]‐Cluster of HydF is not Required for the Binding and Transfer of the Diiron Site of [FeFe]‐Hydrogenases

Abstract

The active site of [FeFe]‐hydrogenases contains a cubane [4Fe‐4S]‐cluster and a unique diiron cluster with biologically unusual CO and CN− ligands. The biogenesis of this diiron site, termed [2FeH], requires the maturation proteins HydE, HydF and HydG. During the maturation process HydF serves as a scaffold protein for the final assembly steps and the subsequent transfer of the [2FeH] precursor, termed [2FeP], to the [FeFe]‐hydrogenase. The binding site of [2FeP] in HydF has not been elucidated, however, the [4Fe‐4S]‐cluster of HydF was considered as a possible binding partner of [2FeP]. By targeting individual amino acids in HydF from Thermosipho melanesiensis using site directed mutagenesis, we examined the postulated binding mechanism as well as the importance and putative involvement of the [4Fe‐4S]‐cluster for binding and transferring [2FeP]. Surprisingly, our results suggest that binding or transfer of [2FeP] does not involve the proposed binding mechanism or the presence of a [4Fe‐4S]‐cluster at all.

Semisynthetic maturation of [FeFe]-hydrogenase using [Fe2(μ-SH)2(CN)2(CO)4]2−: key roles for HydF and GTP

“…However, a recent report showed that synthetic [2Fe] F can bind to HydF and activate HydA even in the absence of the [4Fe–4S] cluster. 39 Further studies are needed to determine the details of how [2Fe] E binds to HydF, and specifically whether [2Fe] E binds directly to the [4Fe–4S] cluster of HydF.…”

A Conserved Binding Pocket in HydF is Essential for Biological Assembly and Coordination of the Diiron Site of [FeFe]-Hydrogenases

“…Although this exchange did not result in significant changes in the FTIR-spectrum, the overall cluster occupancy was moderately reduced to 64%. In addition, the second CN – -ligand is in close distance to the [4Fe-4S]-cluster in our model, which is in agreement with recently published results that suggested a close proximity of one CN – -ligand to the [4Fe-4S]-cluster …”

“…In addition, the second CN − -ligand is in close distance to the [4Fe-4S]-cluster in our model, which is in agreement with recently published results that suggested a close proximity of one CN − -ligand to the [4Fe-4S]-cluster. 33 Located on the other site of the cavity and positioned toward the adt-bridge of [2Fe] F , there is a highly conserved DIG-motif (conservation degree: D: 100%, I: 100% and G: 93). The high conservation degree and the surface exposed location of these residues suit a possible role during adt synthesis or its coordination.…”

“… − [2Fe] F resembles the final [2Fe] H site in HydA, only differing in ligand number and configuration, which is adopted after its transfer to HydA by HydF. HydF is a homodimeric protein with GTPase activity, the function of which has recently been suggested to be important for the final assembly of the adt-bridge. ,− Each HydF monomer contains a [4Fe-4S]-cluster which was postulated to be necessary for binding [2Fe] F via one of its CN – -ligands. ,, However, we have demonstrated that the [4Fe-4S]-cluster is not necessarily required for binding [2Fe] F , questioning the postulated binding mode of the precursor to HydF …”

“…Fourier-transform-infrared (FTIR)-spectroscopy was used to monitor the presence or absence of bound [2Fe] F in each variant either after loading them with an artificial [2Fe] F mimic ([2Fe] MIM ) or after coexpression with HydE and HydG. H 2 production assays with HydF were conducted to indirectly follow the activation of HydA from Chlamydomonas reinhardtii ( Cr HydA1) as shown previously …”